Sequential accumulation of dynein and its regulatory proteins at the spindle region in the Caenorhabditis elegans embryo

Cytoplasmic dynein is responsible for various cellular processes during the cell cycle. The mechanism by which its activity is regulated spatially and temporarily inside the cell remains elusive. There are various regulatory proteins of dynein, including dynactin, NDEL1/NUD-2, and LIS1. Characterizing the spatiotemporal localization of regulatory proteins in vivo will aid understanding of the cellular regulation of dynein. Here, we focused on spindle formation in the Caenorhabditis elegans early embryo, wherein dynein and its regulatory proteins translocated from the cytoplasm to the spindle region upon nuclear envelope breakdown (NEBD). We found that (i) a limited set of dynein regulatory proteins accumulated in the spindle region, (ii) the spatial localization patterns were distinct among the regulators, and (iii) the regulatory proteins did not accumulate in the spindle region simultaneously but sequentially. Furthermore, the accumulation of NUD-2 was unique among the regulators. NUD-2 started to accumulate before NEBD (pre-NEBD accumulation), and exhibited the highest enrichment compared to the cytoplasmic concentration. Using a protein injection approach, we revealed that the C-terminal helix of NUD-2 was responsible for pre-NEBD accumulation. These findings suggest a fine temporal control of the subcellular localization of regulatory proteins.


Molecular weight is not the determinant of accumulation order
We observed that the timing of accumulation differed between dynein and its regulatory proteins. The proteins were expected to enter the spindle region mainly through diffusion because NPCs, which act as a diffusion barrier and as a mediator for active nucleocytoplasmic transport, underwent disassembly by that time 1 . Thus, we hypothesized that the difference in diffusion rate depending on molecular weight accounted for the temporal difference. This hypothesis was supported by the fact that the accumulation order of dynein-regulatory proteins coincided with the order of molecular weights; NUD-2 dimer (~69 kDa) accumulated first, followed by LIS-1 dimer (92 kDa) and LIN-5 dimer (187 kDa), with final accumulation of dynactin (~1.0 M) and dynein (1.4 M) (Figure 4a). To examine the effect of molecular weight on the accumulation, we observed the temporal dynamics of polymers with defined molecular sizes using an injection method ( Figure S4a Although previous studies have reported the presence of injected dextran in interphase embryos 2,3 , it was unclear whether they accumulated at the spindle region during mitosis. Thus, we decided to observe the accumulation events of polyethylene glycol (PEG) as well as dextran. By observing dextran (40 kDa) and PEG (40 kDa) accumulations, we found that dextran showed NEBDdependent accumulation in the spindle region ( Figure S4b), while PEG was excluded from the nucleus throughout the cell cycle ( Figures S4c and S4d). Notably, PEG with a smaller molecular weight (5 kDa), which was expected to be below the diffusion limit of NPCs, was also excluded from the nucleus ( Figure S4e), suggesting that the event of accumulation of a polymer at the spindle region was dependent on physicochemical properties, such as the existence of branching in the polymer structure.
We then compared the accumulation dynamics of dextrans with molecular weights of 3, 40, 70, and 150 k. Dextran (3 kDa) presented with continuous accumulation in the nuclear region throughout the cell cycle ( Figure S4f and S4g), probably because the molecular weight was below the diffusion threshold of the nuclear pore complex. Other dextrans exhibited NEBD-dependent accumulation at the nascent spindle region ( Figure S4b and Movie S4). As depicted in the time series of the normalized NI, dextrans accumulated only after NEBD ( Figure S4g). The time series also did not demonstrate any marked difference in the timing of dextran accumulation. This result indicated that molecular weight was not a determinant factor for the accumulation order.
Although molecular weight was not deemed the determinant, it was notable that exogenous polymers showed an accumulation pattern similar to that shown by dynein and the regulatory proteins.
Additionally, it was observed that the proteins, dynein and dynactin, mainly accumulated through the establishment of interaction with microtubules (Figures 3d and 3e). Thus, we examined whether the accumulation of dextran depended on the interaction with microtubules. The observation of dextran in the nocodazole-treated embryos showed that it continued to accumulate at the nascent spindle region ( Figure S4h), indicating that the accumulation of dextran was not dependent on microtubules such as LIS-1, NUD-2, and LIN-5 (Figures 3a-c). Furthermore, similar to LIN-5, dextrans showed a uniform distribution in the nascent spindle region ( Figure S4i). Although the accumulation dynamics of dextrans shared several characteristics with dynein and the regulatory proteins, dextran did not present with accumulation before NEBD, as that observed for NUD-2, suggesting an additional requirement for such an accumulation pattern.

Pre-NEBD accumulation of NUD-2 is independent of NEBD
Among the proteins observed, NUD-2 showed a distinct accumulation pattern compared with the other proteins; accumulation started approximately 4 min before NEBD and the highest maximum normalized NI of approximately 4.5-fold was noted (Figures 2d and 4a). We termed this phenomenon "pre-NEBD accumulation" and investigated it comprehensively.
We observed that the initiation time of pre-NEBD accumulation was around the time of the pronuclear meeting. If the pre-NEBD accumulation is dependent on pronuclear meetings, it should occur only at the 1-cell stage because the pronuclear meeting is specific to the 1-cell stage. However, this was not the case. We found that pre-NEBD accumulation also occurred in the later stage embryos (2-16-cell stage; Figure S6a). Interestingly, as development proceeded, the degree of accumulation through pre-NEBD accumulation increased, while the final normalized NI after post-NEBD accumulation did not vary among the cell stages ( Figures S6b-d). In contrast to the early embryos, in oocytes, NUD-2 did not accumulate to the nuclear region prior to the NEBD of the oocyte meiosis.
The post-NEBD accumulation was observed for the oocyte meiosis ( Figures S6e and S6f). Moreover, we found that NUD-2 localized at the nuclear membranes in all oocytes except the most proximal (-1) one ( Figure S6e), in contrast to the early embryos. These results suggest that pre-NEBD accumulation is specific to mitotic division, whereas post-NEBD accumulation is universal to mitosis and meiosis.
We then investigated the relationship between pre-NEBD accumulation of NUD-2 and NEBD.
We focused on a key aspect: was pre-NEBD accumulation coupled with NEBD? If pre-NEBD accumulation depends on NEBD, the timing of pre-NEBD accumulation between sperm-and oocytederived pronuclei will differ in the presence of nocodazole. Nocodazole treatment impairs pronuclear meeting, which in turn delays NEBD of the oocyte pronucleus due to the lack of signals from centrosomes attached to the sperm pronucleus 4-6 . When NEBD of oocyte pronucleus was delayed, there was no delay in the initiation time of pre-NEBD accumulation and it occurred at the same time as that of sperm pronucleus ( Figures S6g and S6h). After reaching a value of approximately 1.3, the normalized NI of the oocyte pronuclei showed the achievement of a steady state for several minutes, while the normalized NI of the sperm pronucleus showed a transition to post-NEBD accumulation.
These results suggest that pre-NEBD accumulation is a distinct process from the post-NEBD accumulation and is regulated by factors independent of NEBD.

NUD-2 exhibits a distinct accumulation pathway from tubulin
Ran, a small GTPase protein, plays a central role in nuclear transport. A recent study revealed that Ran contributed to the accumulation of a tubulin chaperone in the nuclear region before NEBD in Drosophila melanogaster 7 . We have previously shown that RAN-1 is necessary for the post-NEBD accumulation of tubulin in C. elegans embryos 8 . We sought to ascertain whether Ran was involved in the pre-NEBD accumulation of NUD-2 by conducting knockdown experiments for C. elegans Ran, ran-1. In the ran-1 (RNAi) embryos, we confirmed a reduction in cell size, nuclear size, and observed defects in mitosis ( Figure S7a), as those previously described [9][10][11] . The defect in cytokinesis maintained These results suggest that NUD-2 exhibits a different accumulation pathway from tubulin, whose post-NEBD accumulation is dependent on RAN-1 8 .
To investigate the details of NUD-2 accumulation in the ran-1 (RNAi) embryos, we analyzed the time series of the normalized NI. In ran-1 (RNAi) embryos, we could not determine the timing of NEBD from the localization pattern of histones, and thus it was difficult to differentiate between pre-NEBD and post-NEBD accumulation events of NUD-2. As shown in Figure S7e, NUD-2 signals increased at an approximately constant rate. This increasing pattern was somewhat different from the unperturbed condition where we observed slower accumulation followed by a short constant phase before NEBD and faster accumulation after NEBD (Figure 4a). We considered that either pre-or post-NEBD accumulation was impaired by ran-1 (RNAi). By comparing the rate of accumulation, we found that the accumulation rate under the ran-1 (RNAi) condition was more similar to that under the unperturbed condition ( Figure S7e). Furthermore, the maximum normalized NI of NUD-2 in the absence of RAN-1 was estimated to be 3.8 ± 1.6 (based on 7 increase events in 5 embryos), comparable to that of post-NEBD accumulations under the unperturbed condition (4.7 ± 0.6). These results suggest that Ran is necessary for pre-NEBD accumulation, but is not vital in the post-NEBD accumulation of NUD-2. This is in contrast to tubulin, where post-NEBD accumulation is impaired by ran-1 (RNAi) 8 . These results suggest that the mechanism of post-NEBD accumulation is different between NUD-2 and tubulin.                        This work